Cichlids of the tribe Heroini have long been a source of taxonomical conflict. In
particular, the species included in the Herichthys bartoni group
have failed to be recovered as monophyletic in different molecular studies. In this
paper we use traditional and geometric morphometrics to evaluate morphological
variation in the species included in the H. bartoni complex in order
to evaluate the number of species it contains. An update of a previously published
DNA barcoding study suggests the existence of three genetic clusters that included
the six recognized species analyzed in this study, none of them recovered as
monophyletic. On the other hand, geometric morphometrics arise as a useful tool to
discriminate species due that traditional morphometrics showed a high overlap in the
characters analyzed that prevents the proposal of diagnostic characters.

Aquatic environments can show great spatial and temporal variations in both biotic and
abiotic parameters. In these environments, many fish species show extreme morphological
differences between highly contrasting habitats (Langerhans et al., 2003). Cichlids in particular are known
for their spectacular adaptive radiation and high phenotypic plasticity, which makes
this group an excellent model for ecological and evolutionary studies (Klingenberg et al., 2003). In the
recent years, these high levels of variation have translated into a very unstable
taxonomy, which has led to several nomenclatural changes and unclear boundaries among
species. Originally, the genus Herichthys comprised four species that
inhabit the coastal basins of the Gulf of Mexico from Texas to Veracruz (Miller, 1966). Later, Kullander (1996) included in Herichthys five
species that had previously been in the genus Cichlasoma, and De la Maza-Benignos & Lozano-Vilano (2013)
recently described three new species to complete the 12 currently recognized species.
Recent phylogenetic studies have confirmed the monophyly of the genus but not of the
species included in it (Hulsey et al.,
2004; Concheiro-Pérez et al.,
2007; Říčan et al.,
2008; Říčan et al.,
2013) because the two different haplotypes of H. labridens
were recovered as polyphyletic. In addition, these works suggested the
existence of two groups of species that were corroborated with the use of other
molecular markers (Říčan et al.,
2008; Mejía et al.,
2012; Říčan et al.,
2013) and morphometric characters.

(De la Maza-Benignos & Lozano-Vilano, 2013).
One group of species was associated with H. cyanogutattus: H.
cyanogutattus, H. deppii, H.
minckleyi, H. carpintis, and H.
tamasopoensis, and the other group was associated with H.
bartoni: H. bartoni, H. steindachneri,
H. pantostictus, H. labridens, H.
pame, H. pratinus, and H. molango.
Finally, a recent DNA barcoding study that included samples of the complete geographic
distribution of the H. bartoni species group (64 individuals from 22
localities) defined three well-supported phylogenetic groups (León-Romero et al., 2012). Phylogenetic group I
included haplotypes of H. bartoni and H. labridens
from San Luis Potosí, phylogenetic group II included all of the haplotypes of H.
steindachneri (the only species found to be monophyletic), and phylogenetic
group III included haplotypes of H. pantostictus and H.
labridens from the state of Hidalgo (León-Romero et al., 2012). All of the molecular studies
mentioned above support the earlier suggestions of Taylor & Miller (1983) and Miller
et al. (2005) that H. labridens might
comprise two different species. In fact, in the recent revision of the species performed
by De la Maza-Benignos & Lozano-Vilano
(2013), they restrict the distribution of H. labridens to Laguna
Media Luna and the headwaters of río Verde, San Luis Potosí, and describe three of the
populations that were previously described as H. labridens as
H. pratinus endemic to río el Salto, San Luis Potosí; H.
pame (endemic to Rio Gallinas and its tributaries) and H.
molango (endemic to Laguna Atezca in the state of Hidalgo Mexico) (Fig. 1). Nevertheless, in the geographic distribution
of the H. bartoni species group, there are several localities that are
currently identified as H. labridens (marked with a cross) that remain
taxonomically uncertain. Moreover, though De la
Maza-Benignos & Lozano-Vilano (2013) consider the existence of diagnostic
characters for each species, it is clear in their summary Tables 4 and 5 that there are several overlaps among the species for
all of the characters that they analyzed. In this study, we performed a morphological
revision of the Herichthys bartoni species group using meristic,
morphometric, and geometric morphometrics data to assess the proposal of recently
described species and the identity of uncertain populations. Following the proposal of
Miller et al. (2005), we
restricted the name of H. labridens to the populations of the
headwaters of the río Verde, San Luis Potosí and referred to the rest of populations
that are currently identified as H. labridens and included in
phylogenetic group III, as defined by León-Romero
et al. (2012), as H. cf.
labridens, except for H. pame and H.
molango, which were considered distinct species. Finally, we rejected the
recent proposal of De la Maza-Benignos et al. (2014), who suggested
that the species included in the H. labridens species group must be
segregated into a new genus named Nosferatu because the morphological
characters that support both genera are also present in species of the other genus
(Pérez-Miranda et al. in prep.).

Figure 1. Geographic distribution of the species included in the Herichthys bartoni
group. Note: The species H. pratinus was not included in this work.

Table 1. Matrix of classification for the seven species of the Herichthys bartoni
species group derived from the Discriminant Function Analysis (DFA). The data
are presented as percents of correct classification for each data set.

Species

Meristic

Mossimann

Proportions

H. bartoni

92,1053

86,8421

87,1795

H. cf. labridens

92,8571

93,8111

93,8312

H. labridens

20,5480

64,3836

65,7534

H. molango

0,0000

0,0000

39,1304

H. pame

0,0000

4,7619

0,0000

H. pantostictum

30,2326

67,4419

81,3954

H. steindachneri

50,0000

29,6296

46,6667

Total

67,5183

79,1423

80,8429

Table 2. Procrustes distances among the seven taxa of the the Herichthys bartoni
species group included in this study. Above the diagonal the results for the 15
landmarks of the head, below the diagonal the results for the 25 landmarks of
the body. *denotes significant p values (p< 0.05) after 10,000
iterations.

H. bartoni

H. labridens

H. steindachneri

H. cf. labridens

H. pantostictus

H. pame

H. molango

H. bartoni

0.041*

0.0489*

0.0355*

0.0552*

0.0602*

0,0624

H. labridens

0.0337*

0,0232

0.0176*

0.0430*

0,0406

0,0783

H. steindachneri

0.0394*

0.0236*

0.0250*

0,0433

*

0,0771

H.cf. labridens

0.0348*

0.0126*

0.0227*

0.0552*

0.0602*

0,0624

H. pantostictus

0.0380*

0.0162*

0.0212*

0.013*

0.0615*

0.0947*

H. pame

0.047*

0.0488*

0.0585*

0.0485*

0.0526*

0.0969*

H. molango

0.0603*

0.0393*

0.0414*

0.0421*

0.0424*

0.0635*

Table 3. Descriptive statistics for the 18 morphometric data adjusted as proportions
of the standard length (SL) and nine morphometric data adjusted as proportions
of the head length (HL) used in this study. The mean, minimum, maximum and the
standard deviation of the value for each Herichthys species are expressed as
percentages.

% SL

Phylogenetic group I

Phylogenetic group II

Phylogenetic group III

H. bartoni

H. labridens

H. steindachneri

H. pame

H. pantostictus

H. molango

min

X

max

SD

min

X

max

SD

min

X

max

SD

min

X

max

SD

min

X

max

SD

min

X

max

SD

Total length of the anal fin (LAF)

25

31

37

3

30

35

40

2

29

35

45

4

31

35

38

2

32

36

41

2

27

37

48

3

Total length of the dorsal fin (LDF)

53

61

68

3

51

65

71

3

54

63

75

5

62

65

70

2

58

67

73

3

55

69

82

4

Total length of the dorsal fin of spines (DFE)

39

45

50

2

33

49

57

3

42

49

61

4

45

49

52

2

47

51

56

2

36

50

59

3

Total length of the dorsal fin of rays (DFR)

9

15

22

2

10

17

24

3

8

14

24

3

12

17

21

3

9

16

20

2

11

19

34

3

Total length of the anal fin of spines (AFE)

14

18

24

2

14

20

27

2

13

21

29

4

11

18

21

2

18

22

26

2

9

20

27

3

Total length of the anal fin of rays (AFR)

8

12

17

2

12

15

23

2

10

15

21

2

12

16

22

3

12

14

19

1

5

17

30

3

Total length of the pectoral fin (LPF)

20

23

27

2

20

23

29

2

20

24

29

2

21

23

25

1

19

24

28

2

17

24

30

2

Total length of the pelvic fin (LVF)

16

21

28

2

16

23

29

2

20

23

30

2

19

23

26

2

20

24

27

2

20

25

32

2

Predorsal length (PDL)

33

40

45

3

29

35

40

2

34

39

45

2

30

34

38

2

31

36

40

2

26

35

42

3

Preanal length (PAL)

65

70

74

2

61

64

68

2

59

66

70

2

61

65

68

2

63

67

70

2

49

65

73

3

Length of the caudal peduncle (LCP)

10

13

15

1

10

12

16

1

8

12

17

2

11

12

15

1

10

12

14

1

9

11

14

1

Length of the dorsal fin at its base (LDB)

40

47

54

2

47

51

54

2

42

48

57

4

46

50

53

2

48

52

56

2

24

52

67

4

Length of the anal fin at its base (LAB)

14

16

20

1

13

19

24

2

13

19

24

3

17

19

22

2

18

21

26

2

14

19

27

2

Head length (HLE)

34

38

44

2

31

34

40

2

33

36

41

2

30

33

35

2

30

34

37

2

20

34

39

3

Length of the post ascending premaxillary process (PPP)

21

24

29

2

19

26

30

3

20

24

42

3

24

27

29

1

17

22

34

3

29

27

33

2

Distance between the anal fin and the base of the pelvic fins
(DBF)

22

29

32

2

23

29

33

2

23

28

39

3

27

29

33

2

22

27

32

2

23

29

35

4

Body height (BHE)

37

41

45

2

37

42

46

2

32

39

46

4

38

41

43

2

38

41

45

2

33

41

47

2

Height of the caudal peduncle (HCP)

14

16

17

1

14

16

18

1

12

15

18

1

15

16

18

1

15

16

18

1

13

16

19

1

% HL

Postorbital length (POL)

40

47

53

3

39

47

52

2

35

42

48

3

42

46

51

2

36

44

56

3

30

44

63

3

Length of the upper maxilla (UML)

12

20

29

4

16

22

28

2

13

22

31

3

16

21

27

3

17

22

32

3

16

23

36

3

Length of the lower maxilla (LLM)

13

21

30

4

16

22

28

2

15

26

32

3

15

21

26

3

16

22

32

3

16

23

36

3

Snout length (SNL)

24

30

40

4

26

33

38

2

27

34

35

3

28

37

38

3

23

31

37

3

21

35

47

3

Length of the ascending premaxillary process (LPP)

31

36

43

3

24

41

51

7

30

40

66

6

38

48

53

3

30

38

45

3

27

47

56

4

Head height at the eye (HHE)

68

81

110

8

74

96

119

10

59

80

110

10

85

97

117

8

74

88

110

8

64

93

136

10

Eye diameter (EYD)

20

25

32

3

19

24

30

2

18

25

33

3

18

23

30

2

23

27

32

2

19

25

35

3

Height of the head at the preopercle (HHP)

78

91

120

9

79

110

131

10

62

90

110

11

99

110

130

8

85

99

120

8

87

110

150

9

Intraocular distance (IOD)

23

27

38

4

29

35

42

3

23

30

45

5

31

37

42

3

25

32

39

3

24

35

46

3

Table 4. Descriptive statistics for the 13 meristic data used in this study. The
mode, minimum, maximum and the frequency of the mode for each species are
presented.

Counts

Phylogenetic group I

Phylogenetic group II

Phylogenetic group III

H. bartoni

H. labridens

H. steindachneri

H. pame

H. pantostictus

H. molango

min

m

max

freq

min

m

max

freq

min

m

max

freq

min

m

max

freq

min

m

max

freq

min

m

max

freq

Number of spines in the dorsal fin

14

15

16

66

14

16

17

70

15

16

18

84

15

16

16

76

15

16

17

58

14

16

18

68

Number of rays in the dorsal fin

10

11

12

59

10

11

13

55

9

10

12

48

9

11

12

38

9

11

12

53

9

11

13

68

Number of spines in the anal fin

3

4

5

82

4

5

6

56

4

5

6

66

4

5

6

81

5

6

6

53

4

5

6

69

Number of rays in the anal fin

8

9

10

71

8

9

11

71

8

8

10

57

8

9

10

57

8

9

10

70

7

9

11

68

Number of rays in the pectoral fins

13

14

15

59

13

15

16

57

14

14 & 15

15

50

14

15

15

57

14

15

15

58

13

15

16

68

Number of rays in the pelvic fins

5

5

5

100

4

5

5

97

4

5

5

95

3

5

5

90

5

5

5

100

4

5

5

98

Number of gill rakers in the dorsal arm

2

2

3

61

1

2

4

68

2

2

3

86

2

2

3

90

2

3

4

49

1

2

4

83

Number of gill rakers in the ventral arm

3

5

7

49

4

5

6

56

3

5

6

57

4

5

6

52

4

5

5

98

3

5

7

51

Number of scales in a longitudinal series

25

27

31

46

25

28

30

58

25

26

31

29

23

28

30

62

27

28

29

60

16

27

30

45

Number of circumpeduncular scales

14

16

18

69

14

16

20

52

14

14 & 16

18

48

16

16

18

81

12

16

16

72

14

16

19

59

Number of scales in the first portion of the lateral line

14

18

19

36

15

19

22

41

13

18 & 19

21

34

15

19

20

38

16

20

21

51

8

19

21

37

Number of scales in the second portion of the lateral line

6

9

14

28

5

10 & 11

15

25

5

9

12

32

9

11

20

33

7

10

12

37

3

10

20

34

Total number of scales in the lateral line

24

27

32

31

21

30

34

27

22

27

30

23

26

30

31

48

22

30

31

39

12

29

35

29

Material and Methods

The material consists of 544 individuals from 47 localities that were examined using
meristical and traditional morphometrics analysis and 510 individuals from 48 localities
that were examined using geometric morphometrics analysis (see Appendix). All specimens were identified using the diagnostic
characters described by Taylor & Miller
(1983) and De la Maza-Benignos &
Lozano-Vilano (2013) and are deposited in the Colección Nacional de Peces
Dulceacuícolas Mexicanos de la Escuela Nacional de Ciencias Biológicas (IPN-ENCB-P). In
line with the information presented earlier (Miller
et al., 2005; León-Romero
et al., 2012; De la
Maza-Benignos & Lozano-Vilano, 2013), we include seven taxa: H.
bartoni, H. labridens, H. steindachneri,
H. pantostictus, H. pame, H.
molango, and H. cf. labridens, however, we
cannot include samples from H. pratinus.

Phylogenetic DNA barcoding updated. We generated the DNA barcodings for
three individuals from H. pame to assign a phylogenetic position within
the previously published tree of León-Romero et
al. (2012). The parameters of the Bayesian analysis and the
selected outgroups were the same as the previously published data. We were unable to
incorporate individuals from H. molango because this species was
formalin fixed and preserved.

Traditional morphometrics and meristic data. A total of 29 morphometric
characters were measured in each individual using a digital caliper with a precision of
0.01 mm: total length (TLE), standard length (SLE), total length of the anal fin (LAF),
total length of the dorsal fin (LDF), total length of the dorsal fin of spines (DFE),
total length of the dorsal fin of rays (DFR), total length of the anal fin of spines
(AFE), total length of the anal fin of rays (AFR), total length of the pectoral fin
(LPF), total length of the pelvic fin (LVF), predorsal length (PDL), preanal length
(PAL), postorbital length (POL), length of the upper maxilla (UML), length of the lower
maxilla (LLM), length of the caudal peduncle (LCP), length of the dorsal fin at its base
(LDB), length of the anal fin at its base (LAB), head length (HLE), snout length (SNL),
length of the ascending premaxillary process (LPP), length of the post ascending
premaxillary process (PPP), distance between the anal fin and the base of the pelvic
fins (DBF), body height (BHE), head height at the eye (HHE), height of the caudal
peduncle (HCP), eye diameter (EYD), height of the head at the preopercle (HHP), and
intraocular distance (IOD). These morphometric data were standardized to remove the
effects of size using two different approaches. The traditional approach used in fish
taxonomy consists of estimating the proportions of each variable relative to the
standard length (for all characters) or as a proportion of the head length (for
characters related to this structure); however, this method can be used only if the
growth is isometric. Thus, we used the Mossimann method (Butler & Losos, 2002) to estimate the geometric mean of all variables to
estimate the log (size) for use as an additional variable in statistical analysis. A
total of 13 meristical characters were recorded for each specimen: number of spines in
the dorsal fin, number of rays in the dorsal fin, number of spines in the anal fin,
number of rays in the anal fin, number of rays in the pectoral fins, number of rays in
the pelvic fins, number of gill rakers in the dorsal arm, number of gill rakers in the
ventral arm, number of scales in a longitudinal series, number of circumpeduncular
scales, number of scales in the first portion of the lateral line, number of scales in
the second portion of the lateral line, and total number of scales in the lateral line.
To compare our results with those previously published by De la Maza-Benignos & Lozano-Vilano (2013), we performed a
discriminant function analysis (DFA) for each data set in Statistica 10 (Statsoft Inc.).
Additionally, a multidimensional scaling analysis (MDS) was performed for each data set
using Euclidean distances, as suggested by McMahan et al. (2011). This test was considered
informative if the stress level was lower than 0.15. All analyses were performed in PAST
3.01 (Hammer, Harper & Ryan, 2001). Finally,
to identify significant differences among the seven taxa, a one-way ANOVA was conducted
on the morphometric data adjusted by proportions, and an ANCOVA was conducted on the
morphometric data adjusted using the Mossimann method, with the logsize variable serving
as the covariate. In both cases, Tukey's multiple comparison test was used to identify
significant differences among taxa in Statistica 10. Conversely, to identify significant
differences among the 13 recorded meristic characters, a Kruskal-Wallis analysis using a
multiple comparisons of the mean ranks test was performed and implemented in Statistica
10.

Geometric morphometrics. Each specimen was photographed in the left view,
and 25 landmarks were digitized for the body, as was a subsample of 15 landmarks of the
head, in accordance with Trapani (2003) and Genner et al. (2007) in tpsdig
(Rohlf, 2010) (Fig. 2). To eliminate the effect of curvature caused by the preservation
method, we performed a regression with the "unbend specimens" option in the tpsutil
software (Rohlf, 2012). For the body, we used
four landmarks (2, 9, 18, and 20), and for the head, we used three landmarks (2, 18, and
19). The Bookstein coordinates generated were converted to Procrustes distances using
CoordGen 6, which was included in the program IMP (Sheets et al., 2001). To eliminate the allometric effect
associated with growth, we performed a multivariate regression analysis using the
Procrustes distances as the dependent variable and the size of the centroid as the
independent variable. The adjusted Procrustes distances were used as descriptors of the
level of differences among body and head shapes between the groups; the significance of
the differences was evaluated using a permutation test with 10,000 iterations (Elmer et al., 2010) in MorphoJ
1.03C (Klingenberg, 2011). Finally, the residuals
of the regression analysis were used in a canonical analysis to compare the seven
groups. Similar to the Procrustes distances, the significance of the
differences was evaluated using a permutation test with 10,000 iterations in MorphoJ
1.03C (Klingenberg, 2011).

Figure 2. Landmarks recorded in this study. 1. Anterior end of the lower maxilla 2.
Anterior end of the upper maxilla 3. Length of the ascending premaxillary
process 4. End of the supraoccipital bone 5. Start of the dorsal fin 6. Last
spine of the dorsal fin 7. End of the dorsal fin 8. Upper boundary of the
caudal fin 9. Center of the caudal fin 10. Base of the caudal fin 11. End of
the anal fin 12. Last spine of the anal fin 13. Origin of the anal fin 14.
Origin of the pelvic fin 15. Posterior end of the lower maxilla 16. Posterior
end of the upper lip 17. Maximum point of curvature at the preoperculum 18.
Upper end of the preoperculum 19. Upper end of the operculum 20. Most posterior
end at the operculum 21. Origin of the pectoral fin 22. Upper extreme of the
sphenotic orbit 23. Base of the sphenotic orbit 24. Left extreme of the
sphenotic orbit 25. Right extreme of the sphenotic orbit.

Results

DNA barcoding. The phylogenetic analysis of the mitochondrial COI confirms
the previously published results using COI and other molecular markers that the genus
Herichthys comprises a well-supported monophyletic group (BPP=1.0)
and that this genus includes two well-supported clades: the group of species related to
H. cyanoguttatus (BPP = 1.0) and the group of species related to
H. bartoni analyzed in this study (BPP= 1.0). Contrary to the
previously published results (León-Romero et
al., 2012), none of the species included in the H.
bartoni species group was recovered as monophyletic, due to the unexpected
inclusion of the haplotypes of H. pame in the clade of H.
steindachneri (Fig. 3). However, three
well-supported phylogenetic groups (BPP = 1.0) were recovered, each including a species
that had previously been identified as H. labridens. Phylogenetic group
I includes H. bartoni and H. labridens, phylogenetic
group II includes H. steindachneri and H. pame and
finally, phylogenetic group III includes H. pantostictus and the
individuals considered in this study as H. cf.
labridens, which we synonymize into H. molango (see
below).

Figure 3. Phylogenetic analysis of the Herichthys bartoni species group obtained from
the Bayesian analysis of an approximately 652 bp fragment of the mitochondrial
cytochrome c oxidase subunit I (DNA Barcode). Numbers above nodes are the
Bayesian Posterior Probabilities (BPP) for the principal clades recovered in
the analysis. The haplotypes of the species included in the H. cyanoguttatus
species group were collapsed to facilitate representation.

Traditional morphometric and meristic data. The canonical variance analysis
of the species included in the Herichthys bartoni species group
partially differentiated among H. bartoni, H.
pantostictus, and H. steindachneri for the three data sets
analyzed (Fig. 4); however, the other four species
that were previously described as H. labridens were hardly separated.
These results were confirmed by the classification matrix for the discriminant function
analysis (Table 1). Although the percentage of
corrected classification for the meristic data in H. pantostictus was
only 30.23%, this species is clearly separated from the others when the morphometric
data are adjusted as proportions (81.39%). Conversely, nearly of the 50% of individuals
of H. steindachneri were correctly classified by the meristic data and
the morphometric data adjusted as proportions. Finally, almost 90% of the H.
bartoni individuals were correctly classified in the three data sets. These
results contrast greatly with those found for the other four species. Almost 65% of the
individuals classified as H. labridens from the Media Luna and río
Verde were successfully classified; this value dropped to 40% for H.
molango when the data were adjusted as proportions, as most of the
individuals were classified as H. cf. labridens (data
not shown). It further dropped to only 5% for data that were adjusted using the
Mossimann method for H. pame, in which most of the individuals were
classified as H. labridens (data not shown). Finally, though it is a
widely distributed species, more than 90% of the individuals classified as H.
cf. labridens (=H. molango) were correctly
classified. However, none of the multidimensional scaling analysis performed showed
significant differences because the levels of the stress in the test ranged from 1.415
for the Mossimann adjusted data to 1.471 for the meristic data (data not shown). The
ANOVA revealed that H. bartoni differs from the rest of the species in
six of the characters that are adjusted as proportions (total length of the anal fin
(LAF), total length of the dorsal fin of spines (DFE), total length of the anal fin of
rays (AFR), preanal length (PAL), length of the anal fin at its base (LAB) and
intraocular distance (IOD)), H. molango differs from the rest of the
species in three characters (total length of the anal fin (LAF), total length of the
dorsal fin (LDF) and snout length (SNL)) and H. pantostictus differs
from the rest of the species in four characters (length of the post ascending
premaxillary process (PPP), head height at the eye (HHE), eye diameter (EYD) and height
of the head at the preopercle (HHP)) (Table S1).
Meanwhile, in the ANCOVA for the data adjusted using the Mossimann method, H.
bartoni differs from the rest of the species in the postorbital length
(POL), H. labridens differs from the rest of the species in the
standard length (SLE), height of the head at the preopercle (HHP) and intraocular
distance (IOD) and H. pame differs from the rest of the species in the
total length (TLE), standard length (SLE), total length of the dorsal fin (LDF), total
length of the dorsal fin of spines (DFE), predorsal length (PDL), preanal length (PAL),
length of the caudal peduncle (LCP), length of the ascending premaxillary process (LPP)
and length of the post ascending premaxillary process (PPP) (Table S2). Finally, according to the Kruskal-Wallis H test, only
H. bartoni differs from the rest of the species in number of spines
in the dorsal fin, number of spines in the anal fin, and number of rays in the pectoral
fins (Table S3). Nevertheless, despite the
results of the statistical analysis, a close inspection to the minimum and maximum
values in Tables S1 to S3 indicates that all of
the characters analyzed showed overlapping values that prevented the proposal of
diagnostic characters that would allow us to distinguish any species.

Geometric morphometrics. The analysis of Procrustes distances revealed that
the seven taxa differ significantly in the shape of the body, but only half of the
paired comparisons were significant for the shape of the head (Table 2). Both analysis revealed that the greatest difference in
shapes occurs between H. pame and H. molango
(d=0.0969, P<0.05 for the shape of the head; d=0.0635,
P<0.05 for the shape of the body), while the smallest difference
exists between H. labridens and H. cf.
labridens (d= 0.0176, P<0.05 for the shape of the head;
d=0.0126, P<0.05 for the shape of the body) (Table 2). Conversely, the canonical analysis of the seven groups
failed to reveal a clear pattern in the morphospace for the shape of the head (Fig. 5A-B), but the canonical analysis for the shape
of the body allows the separation of H. bartoni and H.
pame from the rest of the species (Fig.
5C-D).

If we compare the pairs of species included in each of the phylogenetic groups derived
from the DNA barcoding analysis, we can observe that within phylogenetic group I,
H. bartoni (blue dots) and H. labridens (green
dots) have different body shapes. Similar results were found when we compared H.
steindachneri (brown dots) with H. pame (black dots).
However, H. pantostictus (gray dots), H. cf.
labridens (red dots), and H. molango (violet dots) were
barely separated, and a null separation occurred between H. cf.
labridens and H. molango. This information, coupled with
the absence of diagnostic characters, led us to state that the populations included in
this study as H. cf. labridens and H.
molango comprise a single widely distributed species, a result similar to
that found recently by McMahan et al.
(2011) that led them to synonymize Paraneetroplus synspilus
into P. melanurus and led us to synonymize Herichthys
cf. labridens into H. molango.

Diagnosis. Herichthys bartoni can be distinguished from the rest of the species
included in the H. labridens species group by a black-and-white to
light gray coloration in live adult specimens.

Description. Morphometric and meristic data are summarized in Tables 3-4.

Color in life. Body white to light gray, with a series of blotches that
extend from the opercle to the basis of the caudal fin. Dots in the head absent, with
a red to purple axil mark at the pectoral fins.

Remarks. Herichthys bartoni was described originally by
Bean (1892) as Acara
bartoni with four specimens from Hauzteca Potosina (= Huasteca Potosina).
Bean states that the height of the body is contained 2.3 times in the standard
length, the eye diameter is contained 4.5 to 5.5 times in the length of the head and
twice in the length of the snout, the intraocular distance is 66% of the snout length
and the length of the upper maxilla is 40% of the head length. These proportions are
similar to those found in this study (2.2 to 2.7 times the height of the body, 3.1 to
5.1 and 0.81 to 2.0 for the eye diameter and 63% to 127% for the intraocular
distance). The only exception was the 12% to 28% of the upper maxilla compared with
the head length; in fact, none of the specimens reviewed in this study reached such
proportions (the highest value was for one specimen of H. molango
35%), so we think that the Bean (1892)
measurement was taken to the end of the maxillary bone and not to the joint with the
lower maxilla, as we measured. Nevertheless, at least one of the specimens reviewed
by Bean (1892) corresponded to a different
species. Bean states that "In a specimen about 5 inches the cheeks and snout are
profusely covered with minute roundish brown dots", a character present in other
species of the group but not in H. bartoni. The same situation seems
to be true for the material described by Meek
(1904). As noted by De la Maza-Benignos
& Lozano-Vilano (2013) and Meek
(1904) states that the sides of the head are covered with small dark dots
but that in the eight specimens reviewed by Meek
(1904), the number of anal spines was V, a trait observed in only 15% of
the specimens of H. bartoni reviewed in this study. It is thus
likely that the material reviewed by Bean
(1892) and Meek (1904) could
correspond to other species distributed in the Huasteca Potosina, such as H.
pame or H. steindachneri, but not to H.
labridens because the latter species lacks the dots on the head that
occur on H. bartoni.

Diagnosis. There are no unique autapomorphies that allow us to
distinguish Herichthys labridens from the rest of the species of the
group. However, this species could be distinguished from the sympatric species
H. bartoni by its yellow to golden coloration in life and from
the rest of the species of the ensemble by the absence of dots on the head.

Description. Morphometric and meristic data are summarized in Tables 3-4.

Color in life. Body yellow to golden that vanishes to the ventral
region, five to six black blotches that extend from the half of the body to the
caudal fin, red to purple axil mark present.

Color in alcohol. Body brown to reddish-brown, darker at the base of the
dorsal fin. Fins turn to brown or gray, blotches and axil mark could
disappear.

Diagnosis. Herichthys steindachneri could be distinguished from the rest of the
species of the group by its long head, which ranges from 89.6% to 113.2%, in
comparison with the body height at the basis of the pelvic fins.

Description. Morphometric and meristic data are summarized in Tables 3-4.

Color in life. Body greenish-gray, darker in the dorsal to almost white
in the ventral region; a series of blotches that extend from the posterior end of the
eye to the basis of the caudal fin, blotches in the second half of the body could
form up to six bars. Brown or black dots in the head up to basis of the pectoral fins
but not in the dorsal fin, fins yellowish to green, dorsal, anal and caudal usually
with a brown or black blotch at its base; red to purple axil mark present.

Color in alcohol. Body dark gray to light brown, lighter at the ventral
region, with one series of black blotches, fins turn whitish, axial mark could
disappear.

Distribution. Río Gallinas and its tributaries. There are historical
records of its presence in El Pujal and El Rodeo San Luis Potosí and in Villa Aldama
and Jaumave, Tamaulipas.

Remarks. In the recent redescription of the species, De la Maza-Benignos & Lozano-Vilano (2013)
state that this species could be distinguished from the rest of the species by having
the lower jaw extending the upper jaw (prognathous), whereas the opposite condition,
jaws of equal size, are rare and present mostly in juveniles. In the specimens that
we analyzed, the proportion of lower/upper maxilla (prognathous) was barely 1.03;
this condition was present in 54% of the specimens, with no relationship to size (51
mm to 155 mm). Jaws of equal size were present in 46% of the individuals and ranged
from 48 mm to 150 mm in standard length; because of this, we rejected the use of this
trait as a diagnostic characteristic. Conversely, De
la Maza-Benignos & Lozano-Vilano (2013) suggest that the descriptions
of Jordan & Snyder (1900) and Meek (1904)
were based on a composite of H. steindachneri and H.
pame. The proportions found by Jordan & Snyder (1900) were similar to
those found by us. Nevertheless, we found 5 to 9 gill rakers, while in the original
description of H. steindachneri, Jordan & Snyder (1900) found
10, a value similar to that found in H. pame (9-11) by De la Maza-Benignos & Lozano-Vilano (2013).
Meek (1904) description includes V anal
spines, seven anal rays and an upper jaw that is slightly longer than the lower. The
extreme low number of anal rays (7) reported by Meek
(1904) was found only in a single specimen of H. molango
(= H. cf. labridens) reviewed in this study, and an
upper jaw slightly longer than the lower was only found in H. pame
(lower jaw/upper jaw proportion = 0.98). For the former, we concur with De la Maza-Benignos & Lozano-Vilano (2013)
proposal that the descriptions of Jordan & Snyder (1900) and Meek (1904) included two different species.
Contrary to that stated earlier, the geographic distribution of this species includes
not only río Gallinas but also its tributaries, río Tamasopo, río Agua Buena, and río
Ojo Frío in San Luis Potosí. We have historical records of this species in El Pujal
and El Rodeo, the municipality of Río Verde, San Luis Potosí and Villa Aldama and
Jaumave, Tamaulipas. We claim that these specimens were correctly identified.
According to Miller et al.
(2005), H. steindachneri could be distinguished from the
rest of the species by a long head that is usually greater than the body height at
the pelvic fin basis. In the specimens analyzed in this study, the proportion of the
head ranges from 89.6% to 113.2% (X= 96.3%); this proportion was similar in Jaumave,
ranging from 91.5% to 102% (X=94.6%) and Villa Aldama 93.6% to 95.5% (X= 94.7%)
specimens. The head of H. steindachneri is not longer as Miller et al. (2005) suggested,
but it is larger than the head observed in other species. For example, in H.
pame this proportion ranges from 75.3% to 86.1% (X= 81.4%), but only in
the specimens of H. cf. labridens (= H.
molango) from Jaumave does this value range from 79.5% to 85.6%
(X=83.3%), so there is no chance to confuse the two species.

Diagnosis. There are no unique autapomorphies that allow us to
distinguish Herichthys pame from the rest of the species of the
group. Similar to H. steindachneri, the blotches of the body usually
extend to the opercle and to the posterior end of the eye, while in the other
species, the blotches do not extend beyond the opercle. This species could be
distinguished from it sympatric species H. steindachneri by a small
head, which reaches at most 86% of the length in comparison with the height at the
basis of the pelvic fins, in contrast with H. steindachneri, in
which this proportion is greater than 89.6%.

Description. Morphometric and meristic data are summarized in Tables 3-4.

Color in life. Body yellow to light brown, sometimes green, darker in
the dorsal that vanishes to almost white in the ventral region. Dark blotches that
extend from the end of the eye to the caudal fin, blotches in the second half of the
body could form four to seven vertical bars, head with brown or black dots that does
not extend beyond the pectoral fins, pectoral and pelvic fins yellow, while caudal
and anal fin usually light brown or red, in some specimens the dorsal, caudal and
anal fin with speckled, red to purple axil mark present.

Color in alcohol. Body gray to light brown, vanishing to yellow or white
to the ventral region, fins turn whitish, in some specimens, margin of the dorsal fin
turn black, axial mark could disappear.

Distribution. Endemic of río Gallinas and its tributaries.

Remarks. According to the original description, this species could be
separated from the other species of the genus by a small eye diameter (mean 23%, SD
1%) and a long snout (mean 39%, SD 2%), among other traits. Our results indicated
that none of these characters could be diagnostic because a small eye diameter is
also present in H. molango and a long snout is also present in both
H. pantostictus and H. molango. Nevertheless,
this species is clearly separated from the rest in the canonical variable analysis
derived from the analysis of geometric morphometrics of body shape (Fig. 5 ). Herichthys pame was
originally considered a geographic variant of H. labridens by Taylor & Miller (1983, Fig. 4), although Miller
et al. (2005) later reconsidered and state that this
species represents an undescribed species. Although De la Maza-Benignos & Lozano-Vilano (2013) state that this species was
recovered as a sister taxon of H. steindachneri by Hulsey et al. (2004), the DNA
barcoding analysis performed in this study indicates that both species share some
haplotypes and were undistinguished.

Diagnosis. There are no unique autapomorphies that allow us to
distinguish Herichthys pantostictus from the rest of the species in
the group. Most of the specimens of this species showed brown to black dots in the
head and the body, the latter being larger in size, although some populations might
lack this trait. Some specimens of H. molango might also show dots
in the body, but in this species, the dots are disperse and usually red. This species
differs slightly from its sister taxon H. molango in its body shape,
according to geometric morphometrics, and its geographic distribution (see
below).

Description. Morphometric and meristic data are summarized in Tables 3-4.

Color in life. Body gray to pale yellow, sometimes vanishing to white,
dark blotches extending from posterior end of the opercle to the caudal fin,
sometimes forming four to six vertical bars. Head densely covered by brown or black
small dots that extend from the tip of premaxilla to the basis of the dorsal fin.
Most of the specimens with dots in the body that are bigger than those found in the
head, fins black to yellow, dorsal, anal and caudal fin usually with small brown
blotches at its base, red to purple axial mark present.

Distribution. Costal lagoons and rivers in the south of Tamaulipas and
North of Veracruz.

Remarks. According to Taylor &
Miller (1983; Tables 1,3), this species can be distinguished from
H. labridens because the entire body is covered with small dark
brown spots and by longer basal lengths of dorsal and anal fins and a shorter caudal
peduncle. If we compare only the specimens from río Verde and exclude the non-río
Verde specimens from the work of Taylor & Miller
(1983), that could correspond to another species, we can state that in
H. labridens, the dorsal base length ranges from 53.6% to 58.3%,
the anal base length ranges from 21.2% to 24.8% and the caudal peduncle ranges from
14.4% to 17.3%. Conversely, in the original material examined by Taylor & Miller (1983), in H.
pantostictus, the dorsal base length ranges from 54.3% to 60.6%, the anal
base length ranges from 23.3% to 27.1% and the caudal peduncle ranges from 13.5% to
15.6%. The above-mentioned proportions were similar to those found in this study
(Table 3); however, the overlap in the
minimum and maximum values precludes their use as diagnostic characters. In a recent
redescription of the species, De la Maza-Benignos
& Lozano-Vilano (2013) state that in most of the coastal and lagoon
populations, the entire body is covered with dark dots, although some riverine
populations lack this trait; they also state that shallow cheeks and the large eye
diameter allow the differentiation of these species from the rest. Nevertheless, a
close inspection of their table 4 again
reveals a high overlap of these measurements with those from other species, so there
is no diagnostic character that allows the differentiation of H.
pantostictus from the rest of the species. The same is true for the
number and shape of the teeth in the midline of the lower pharyngeal plate. According
to De la Maza-Benignos & Lozano-Vilano
(2013), H. pantostictus can be distinguished from the rest
of the species by having 6 to 7 teeth flanking the midline on each side, conic to
midsize molariform and increasing in side posteriorly. Similar results were found in
the specimens that we reviewed, except the number ranged from 6 to 8 teeth.
Nevertheless, the same pattern was found in the specimens classified in this study
as H. cf. labridens (= H.
molango) from Huejutla, Hidalgo, Tamazunchale, San Luis Potosí and
Jaumave, Tamaulipas and even in H. molango (data not shown). In the
map depicted in Fig. 1, we include a single
locality of H. cf. pantostictus from río Axtla, San
Luis Potosí because this specimen presents dark spots in the body, in the same way,
we assess specimens from the two localities of Jaumave and río Mante, Tamaulipas as
H. cf. labridens (= H. molango)
due to the lack of dark spots on the body. The above facts led us to question the
existence of H. pantostictus and H. molango
(including H. cf. labridens) as two different
species, primarily because of the absence of diagnostic characters. Nevertheless,
despite these findings, in the present study, 81% of the individuals identified as
H. pantostictus were correctly classified in the DFA of the
morphometric data adjusted as proportions (Table
1, Fig. 4). In addition, this species
presents a body shape that is slightly different from that of their sister taxa
H. molango (including H. cf.
labridens) (Fig. 5 ) despite their
shared DNA haplotypes. In such a way, H. pantostictus and H.
molango could be distinguished in part by their geographic distribution,
with the former distributed in South of Tamaulipas and North of Veracruz and the
latter distributed in the South of San Luis Potosí and North of Hidalgo.

Diagnosis. There are no unique autapomorphies that allow us to
distinguish Herichthys molango from the rest of the species of the
group. This species is distinguished from its sister taxon H.
pantostictus primarily by its geographic distribution (see diagnosis of
H. pantostictus above).

Description. Morphometric and meristic data are summarized in Tables 3-4.

Color in life. A highly polymorphic species, body ranges from yellow,
light brown, reddish, gray and almost black, the color vanishes in the ventral region
to pale yellow or almost white. Mid half of the body with irregular black blotches
that extend from opercle to the caudal fin sometimes forming four to six vertical
bands, in some specimens the blotches also in dorsal and anal fin. Some individuals
with red, brown or black disperse dots in the body. Head usually covered with small
brown dots that could be extended to the end of the body but always following the
dorsal fin, red to purple axil mark present.

Color in alcohol. Body dark brown to gray vanishing to the ventral
region, most of the blotches and dots also present, fins turn white, in some
specimens gray or black, axil mark might disappear.

Distribution. Widely distributed in rivers windward of the Sierra Madre
Oriental in the states of Hidalgo and San Luis Potosí.

Remarks. In the original description performed by De la Maza-Benignos & Lozano-Vilano (2013), the authors state
that this species could be distinguished from the rest because it has two rows of 8
to 9 medium-sized molars that flank the midline in the lower pharyngeal plate. This
result contrasts with that found in this study, where the number of teeth was only
seven; additionally, the teeth were short incisives in the front and only molariforms
backward. Similar results were found in other specimens that were previously
classified as H. labridens from the states of Hidalgo and San Luis
Potosí (here named H. cf. labridens= H. molango).
The use of pharyngeal teeth as a diagnostic character is questionable because their
shape could vary with age and diet (Trapani,
2004; Muschick et al.,
2011). In fact, a geometric morphometric analysis in the polymorphic
species H. minckleyi reveals that both morphs (papilliform and
molariform) have a similar shape and represent a single species (Trapani, 2003). Similar results were found in
this study because all of the performed analyses failed to recover significant
differences between H. molango and the specimens included in this
study as H. cf. labridens (now H.
molango). Thus, this is a highly polymorphic species that is
distinguished from its sister taxa H. pantostictus mainly by its
geographic distribution, as H. molango is restricted to the states
of Hidalgo and San Luis Potosí. Contrary to De la
Maza-Benignos & Lozano-Vilano (2013), this species is not distributed
in the río Santa María in the state of Querétaro, Mexico, because the populations
distributed in this area were identified and classified as H. labridens
(see above).

Finally, we want to highlight two important issues that must be evaluated in later
studies. First, when we compare the shapes of the body within each phylogenetic
group, it is clear that the sympatric species in phylogenetic groups I and II showed
greater differences in body shape than did the allopatric species in phylogenetic
group III, which allows us to suggest character displacement. Second, a close
inspection of Figs. 4 and 5 revealed that a similar shape could evolve independently in
different lineages, most likely in response to functional and phylogenetic
constraints. This is the reason that three different species, namely H.
labridens, H. pame, and H. molango,
were previously described as a single species.

Discussion

The DNA barcoding results from this study suggested the existence of three
well-supported phylogenetic groups in the H. bartoni species group:
phylogenetic group I, including haplotypes of H. bartoni and H.
labridens from Media Luna; phylogenetic group II, including haplotypes of
H. steindachneri and H. pame; and phylogenetic
group III, including haplotypes of H. pantostictus and H.
molango (= H. cf. labridens). However, we
were unable to recover a single species as monophyletic. There are several possible
explanations for these results, including maintenance of ancestral polymorphism (Moritz & Cicero 2004) or hybridization and
recent divergence among lineages (Hubert et
al., 2008; Valdez-Moreno
et al., 2009). Recently, Říčan et al. (2013), using seven different molecular
markers, estimate the age of phylogenetic group I, which includes H.
bartoni and H. labridens at approximately 4 my. Conversely,
they state that the other group of species that includes H.
pantostictus, H. steindachneri, and H.
pame have an estimated age of 4.4 my and that the most recent divergence
occurred between H. steindachneri and H. pame only 1.2
my ago. Nevertheless, it is necessary to note that the ages estimated by Říčan et al. (2013) represent the
estimated mean age and are derived from only a single specimen of each species; thus,
according to the 95% HPD depicted in their Fig. 2,
the groups could be older or have had a very recent divergence. Hulsey & García de León (2013), however, suggest the possible
recent introgression (less than 100 years ago) of mtDNA haplotypes (without nuclear DNA
introgression) of H. cyanoguttatus into H. minckleyi
populations based on the presence of the same haplotype of H.
cyanoguttatus from Tamaulipas in Cuatro Cienégas, Coahuila, discarding the
maintenance of ancestral polymorphism. However, an analysis of the DNA barcodes of
H. cyanoguttatus available in Genbank and the BOLD systems suggest
the maintenance of ancestral polymorphism as a result of a species with a very high
effective population size because the same haplotype was recovered in Texas and Central
Mexico (data not shown).

In this work, traditional morphometrics and meristic counts failed to clearly separate
species. Similar results have been found in other cichlids due to the high levels of
overlap between taxonomic characters (Genner et
al., 2007; Schmitter-Soto,
2007; McMahan et al.,
2011; Soria-Barreto et al.,
2011). In recent years, geometric morphometrics have arisen as a useful tool
for discriminating species. For example, Maderbacher
et al. (2008), using 17 landmarks and 9 semi landmarks for
the body, were able to discriminate among three morphs of Tropheus
moorii from Lake Tanganyika, whereas traditional morphometrics failed to
find significant differences because meristic counts and traditional measurements are
very plastic and often overlap between species. Similar results were found by Genner et al. (2007), who used
geometric morphometrics of 25 landmarks from the body coupled with molecular markers and
were able to discriminate between four putative sympatric species of the genus
Diplotaxodon, which had previously been identified only by their
nuptial coloration. However, geometric morphometrics could also have restrictions; for
example, in a recent study of a flock species of the genus Crenicichla
from Uruguay, Burress et al.
(2013) failed to find significant differences among shapes from the 25
landmarks of the body evaluated, although they found significant differences in the
geometric morphometrics of the lower pharyngeal plate. In conclusion, the results of
this study regarding geometric morphometrics allow support for the recent proposal of
De la Maza-Benignos & Lozano-Vilano (2013)
that H. labridens s.l. comprises several species. However, further
studies with other molecular markers are necessary to recover the monophyly of each
species and elucidate the speciation patterns in the H. bartoni species
group.

Acknowledgments

This study was funded by SIP project number 20121320. We also thank to Jorge
Romero-Castillo and Mario Valencia Infante for field assistance, Jon Richey for kindly
review the translated manuscript and an anonymous reviewer for their useful
comments.

References

Bean, T. H. 1892. Notes on fishes collected in Mexico by Prof. Alfredo
Dugès, with descriptions of new species. Proceeding of the United States National
Museum,15: 283-287.
[ Links ]

Kullander, S. 1996. Heroina inonycterina, a new genus and species of
cichlid fish from Western Amazonia, with comments on Cichlasomine systematic.
Ichthyological Exploration of Freshwaters, 7: 149-172.
[ Links ]

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